Fig. 8

Mitochondrial fission 1 protein (Fis1) depletion accompanies mitochondrial respiration dysfunction. a Oxygen consumption rate (OCR) was measured in wild-type (WT) and Fis1 knockout (KO) HeLa cells. Dashed vertical lines indicate the addition of 1 µM oligomycin (Olig), 0.5 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and 1 µM rotenone plus 1 µM antimycin A (Rot/Ant), respectively (n = 8). b Individual parameters for basal respiration and maximal respiration analysis of a. **P < 0.01, *P < 0.05 (two-tailed unpaired Student’s t test, n = 3). c Glucose or galactose-cultured Fis1 KO HeLa cells were transiently transfected with green fluorescent protein (GFP)-tagged vector or syntaxin 17 (STX17) for 24 h. Cells were fixed and immunostained with anti-Tim23 (red) and anti-LC3 (cyan) antibodies. Hoechst, blue. Scale bar, 10 µm. d Quantification of c. ***P < 0.001 (two-tailed unpaired Student’s t test). n = 150 GFP-positive cells/group from three replicates. e Plasmids as indicated were transfected into Fis1 KO HeLa cells. At 6 h post-transfection, cells were cultured in medium with glucose or galactose for further 18 h. Cells were extracted and co-immunoprecipitated with anti-Flag beads, followed by immunoblotting with respective antibodies. The fold change of the interaction between ATG14 and STX17 is shown as mean ± SD from three independent experiments. f GFP-tagged STX17 was transfected into Fis1 KO HeLa cells for 6 h. Then, cells were cultured in medium with glucose or galactose for further 18 h. Immunoblotting of subcellular fractions were analyzed. PNS, post-nuclear supernatant; CYTO, cytosol; ER, endoplasmic reticulum; MAM, mitochondria-associated membranes; MITO, mitochondria. g, h Quantification of relative ratio of GFP-tagged STX17 (g) or endogenous ATG14 (h) in the respective subcellular fractions. *P < 0.05 (two-tailed unpaired Student’s t test, n = 3). Error bars represent SD (a, b, g and h)